2002 Fiscal Year Final Research Report Summary
Development of Inducible Gene Targeting in Mice
| Project/Area Number |
13680906
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Laboratory animal science
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| Research Institution | Tohoku University |
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Principal Investigator |
TAKANO Hiroshi Tohoku University, Graduate School of Medicine, Research Associate, 大学院・医学系研究科, 助手 (00241555)
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| Co-Investigator(Kenkyū-buntansha) |
SUGAWARA Minoru Tohoku University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (20311558)
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| Project Period (FY) |
2001 – 2002
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| Keywords | mouse / inducible gene targeting / Cre recombinase / tamoxifen / mutated estrogen receptr / PCNA |
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| Research Abstract |
To establish an inducible gene targeting system, which relies on a Cre/lox-based strategy making use of a tarnoxifen-inducible Cre recombinase, we have introduced a Cre-ERT2 cDNA into the mouse PCNA locus, allowing to induce modification of gene activity in somatic cells of mouse.
A genomic fragment corresponding to intron 1 of mouse PCNA gene was amplified from mouse genomic DNA by PCR and was used as a probe to obtain mouse PCNA genomic clones from a mouse J1 ES cell genomic library. cDNA of Cre-ERT2 was introduced in frame after the endogenous ATG start coden in exon 1 of PCNA gene and was followed by a mc1neo cassette flanked by loxP sequences. Alternatively, the IRES-Cre-ERT2 was used to construct another vector. Both constructs contained a 1.3kb ApaI-Xbal fragment from the region downstream of exon1 and an 7.2kb fragment from the region upstream of the endogenous ATG start coden of PCNA gene.
Targeting vectors were electroporated into the J1 ES cell line and 295 colonies resistant to G418 were isolated. Thirty-four out of 64 clones tested were identified as homologous recombinat by Southern blot analysis.
We, therefore, have established the PCNA-Cre-ERT2 knock-in ES cell lines. We are at present producing chimeras by ES cell injection into C57bl/6 blastocysts.
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